Sphingosine-1-Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

Production of sphingosine‐1‐phosphate (S1P) is linked to 17β‐estradiol (E2) activity in many estrogen‐responsive cells; in bone development, the role of S1P is unclear. We studied effects of S1P on proliferation and differentiation of human osteoblasts (hOB). Ten nM E2, 1 μM S1P, or 1 μM of the S1P receptor 1 (S1PR1) agonist SEW2871 increased hOB proliferation at 24 hours. S1PR 1, 2, and 3 mRNAs are expressed by hOB but not S1PR4 or S1PR5. Expression of S1PR2 was increased at 7 and 14 days of differentiation, in correspondence with osteoblast‐related mRNAs. Expression of S1PR1 was increased by E2 or S1P in proliferating hOB, whereas S1PR2 mRNA was unaffected in proliferating cells; S1PR3 was not affected by E2 or S1P. Inhibiting sphingosine kinase (SPHK) activity with sphingosine kinase inhibitor (Ski) greatly reduced the E2 proliferative effect. Both E2 and S1P increased SPHK mRNA at 24 hours in hOB. S1P promoted osteoblast proliferation via activating MAP kinase activity. Either E2 or S1P increased S1P synthesis in a fluorescent S1P assay. Interaction of E2 and S1P signaling was indicated by upregulation of E2 receptor mRNA after S1P treatment. E2 and S1P also promoted alkaline phosphatase expression. During osteoblast differentiation, S1P increased bone‐specific mRNAs, similarly to the effects of E2. However, E2 and S1P showed differences in the activation of some osteoblast pathways. Pathway analysis by gene expression arrays was consistent with regulation of pathways of osteoblast differentiation; collagen and cell adhesion proteins centered on Rho/Rac small GTPase signaling and Map kinase or signal transducer and activator of transcription (Stat) intermediates. Transcriptional activation also included significant increases in superoxide dismutase 1 and 2 transcription by either S1P or E2. We demonstrate that the SPHK system is a co‐mediator for osteoblast proliferation and differentiation, which is mainly, but not entirely, complementary to E2, whose effects are mediated by S1PR1 and S1PR2. © 2018 The Authors JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research

Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations

The dose-delivery schedule of conventional chemotherapy, which determines its efficacy and toxicity, is based on the maximum tolerated dose. This strategy has lead to cure and disease control in a significant number of patients but is associated with significant short-term and long-term toxicity. Recent data demonstrate that moderately low-dose chemotherapy may be efficiently combined with immunotherapy, particularly with dendritic cell (DC) vaccines, to improve the overall therapeutic efficacy. However, the direct effects of low and ultra-low concentrations on DCs are still unknown. Here we characterized the effects of low noncytotoxic concentrations of different classes of chemotherapeutic agents on human DCs in vitro. DCs treated with antimicrotubule agents vincristine, vinblastine, and paclitaxel or with antimetabolites 5-aza-2-deoxycytidine and methotrexate, showed increased expression of CD83 and CD40 molecules. Expression of CD80 on DCs was also stimulated by vinblastine, paclitaxel, azacytidine, methotrexate, and mitomycin C used in low nontoxic concentrations. Furthermore, 5-aza-2-deoxycytidine, methotrexate, and mitomycin C increased the ability of human DCs to stimulate proliferation of allogeneic T lymphocytes. Thus, our data demonstrate for the first time that in low noncytotoxic concentrations chemotherapeutic agents do not induce apoptosis of DCs, but directly enhance DC maturation and function. This suggests that modulation of human DCs by noncytotoxic concentrations of antineoplastic drugs, i.e. chemomodulation, might represent a novel approach for up-regulation of functional activity of resident DCs in the tumor microenvironment or improving the efficacy of DCs prepared ex vivo for subsequent vaccinations

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Hydrogeologicke posouzeni - lom Slavetice.

Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Plavebni komora vodniho dila v Brandyse nad Labem.

Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Adrenocorticotropic hormone and 1,25-dihydroxyvitamin D3 enhance human osteogenesis in vitro by synergistically accelerating the expression of bone- specific genes

To improve definition of the physical and hormonal support of bone formation, we studied differentiation of human osteoblasts in vitro at varying combinations of ACTH, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D), and extracellular calcium, with and without added cortisol. Bone mineralization, alkaline phosphatase activity, and osteoblast-specific markers RunX2, osterix, and collagen I increased with 10 pM ACTH, 10 nM 1,25(OH)2D, or at 2mM calcium with important synergistic activity of combinations of any of these stimuli. Signals induced by ACTH at 10–30 min included cAMP, TGF-β, and Erk1/2 phosphorylation. Affymetrix gene expression analysis showed that 2 h treatment of ACTH or 1,25(OH)2D increased the expression of bone regulating and structural mRNAs, including collagen I, biglycan, the vitamin D receptor, and TGF-β. Accelerating expression of these bone-specific genes was confirmed by quantitative PCR. Expression of 1,25(OH)2D 1α-hydroxylase (1α-hydroxylase) increased with 1,25(OH)2D, ACTH, and extracellular calcium from 0.5 to 2 mM. Unlike renal 1α-hydroxylase, in osteoblasts, 1α-hydroxylase activity is independent of parathyroid hormone. In keeping with calcium responsivity, calcium-sensing receptor RNA and protein increased with 10 nM ACTH or 1,25(OH)2D. Inclusion of 200 nM cortisol or 10 nM ACTH in differentiation media blunted osteoblasts alkaline phosphatase response to 1,25(OH)2D and calcium. Our results point to the importance of ACTH in bone maintenance and that extra skeletal (renal) 1,25(OH)2D is required for bone mineralization despite 1α-hydroxylase expression by osteoblasts

Znalecky posudek k hydrogeologicke a inzenyrskogeologicke problematice zalozeni kostela Most - Dekansky kostel.

Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Follicle stimulating hormone receptor in mesenchymal stem cells integrates effects of glycoprotein reproductive hormones

Previously we reported that follicle stimulating hormone (FSH) affects bone degradation in human cells and in FSH-R null mice. Here we describe a FSH-R knockout bone formation phenotype. We used mesenchymal stem cells (MSCs), osteoblast precursors that express follicle stimulating hormone receptor (FSH-R), to determine whether FSH regulates bone formation. FSH stimulates MSC cell adhesion 1–3 h and proliferation at 24 h after addition. On the basis of phylogenetic and clinical precedents, we also examined effects of pregnant levels of human chorionic gonadotropin (hCG) on MSCs. We found effects similar to those of FSH, and RNAi knockdown of FSH-R abrogated both FSH and hCG effects on MSCs. In contrast to effects on MSCs, neither FSH nor hCG had significant effects on osteoblast maturation. Also in MSCs, short term treatment by FSH and hCG altered signaling pathways for proliferation, including Erk1/2 phosphorylation. Our results show augmentation of MSC proliferation by either FSH at menopausal levels or hCG at normal pregnant levels. We conclude that FSH-R participates in regulation of MSC precursor pools in response to either FSH or hCG, integrating the effects of these two glycoprotein hormones